Mechanism investigation of platelet apoptosis inhibition by N-Arachidonoylethanolamine

Abstract

To investigate the mechanism of N- Arachidonoylethanolamine (ANA) on inhibiting platelets (PLT) apoptosis under standard blood bank storage conditions. Samples taken from collected apheresis PLT by the Amicus instrument were split into three parts. An aliquot of 0.5 μmol/L ANA were added to one part of storage PLT as the ANA group; an aliquot of 0.5 μmol/L ANA and 1 μmol/L SR141716 was added to the another part as the ANA + SR141716 group; and the third part without ANA and SR141716 as the control group. These samples were stored on a flat-bed shaker at (22 ± 2) ⁰C for 7 days. The expression of phosphatidyl serine (PS) positive, phospho (p)-Akt, Akt, p-Bad, Bad, caspase-3, caspase-9, cytochrome C (Cyt-C) and BCL-XL interaction with Bak were detected. The rate of PLT PS positive in ANA group decreased significantly than that in control group[ (8.29 ± 1.44) % vs (14.24 ± 2.47) %, P<0.05]. The release of Cyt-C from mitochondria to cytosol in ANA group decreased significantly compared with control group[ (3.29 ± 1.44) % vs (15.24 ± 3.40) %, P<0.05]. Also the expressions of p-Akt and p-Bad in ANA group increased significantly than those in control group[ (71.33 ± 10.26) % vs (35.00 ± 6.00) %, P<0.05; (39.00 ± 9.64) % vs (10.33 ± 1.53) %, P<0.05, respectively]. Higher amounts of Bak protein were co-precipitated with BCL-XL in ANA group than that in control group (about 2.6 fold, P<0.05). The expressions of cleaved caspase- 9 and caspase- 3 in ANA group decreased significantly than those in control group[ (9.63 ± 1.47) % vs (23.24 ± 2.47) %, P<0.05; (6.30 ± 1.40) % vs (13.20 ± 2.50) %, P<0.05, respectively]. There were no significantly changes between ANA+SR141716 and control groups (P>0.05). ANA protected PLTs from apoptosis as a result of inhibiting the release of Cyt-C from mitochondria to cytosol by modifying the expressions of apoptosis-relative proteins.